Fluorescent proteins are proteins that absorb electromagnetic radiation of a particular wavelength and emit electromagnetic radiation of a different longer wavelength. The marine organisms that express fluorescent proteins are predominantly within the phylum Cnidaria, and are estimated to have evolved over 700 million years ago, before organisms of the phylum Cnidaria and the bilateria separated (Shagin et al. (2004), Mol. Biol. Evol. 21, pp. 841-850). Fluorescent proteins exhibit a wide diversity of excitation/emission spectra that extend from cyan to far red, but are generally grouped according to four basic colors: three fluorescent colors (cyan, green, and red) and a non-fluorescent color (purple-blue) (Kelmanson and Matz (2003), Mol. Biol. Evol. 20, pp. 1125-1133). Single organisms have been shown to express multiple fluorescent protein genes, to emit a variety of fluorescent colors (Kelmanson and Matz (2003), Mol. Biol. Evol. 20, pp. 1125-1133; Kao et al. (2007), Mar. Biotechnol. (NY) 9, pp. 733-746), and to express fluorescent proteins in distinct anatomical patterns (Gruber et al. (2008), Biol. Bull. 215, pp. 143-154).
The identification and isolation of fluorescent proteins in various organisms, including marine organisms, has provided a valuable tool to molecular biology. The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria (A. victoria), for example, has become a commonly used reporter molecule for examining various cellular processes, including the regulation of gene expression, the localization and interactions of cellular proteins, the pH of intracellular compartments, and the activities of enzymes (see, e.g., U.S. Pat. Nos. 5,491,084, 5,777,079, and 7,329,735).
The usefulness of A. victoria GFP has led to the identification of numerous other fluorescent proteins, such as fluorescent proteins with emission wavelengths or brightness different from that of GFP. In addition, spectral variants of A. victoria GFP have been disclosed that are excited or emit at wavelengths, for different periods of time, and under different conditions in comparison to the respective properties of native GFP.
Although a number of fluorescent proteins have been disclosed, there still exists a need for fluorescent proteins that exhibit unique biochemical properties. For example, a fluorescent protein that fluoresces with a greater intensity than those previously disclosed would be beneficial, inter alia, in the fields of molecular biology, biochemistry, and drug discovery. Additionally, fluorescent proteins that can detect the interaction of specific molecules and that can track the intra- and intercellular movements of specific molecules would be beneficial, inter alia, in the fields of molecular biology, biochemistry, and drug discovery.
A hallmark of cancer is the imbalance between protein kinase and phosphatase activity. In many cases, overactive protein kinases drive the uncontrolled proliferation of tumors. Akt1 kinase is a well studied kinase that promotes angiogenesis and the development of new blood vessels that feed the uncontrolled growth of solid tumors. Akt1 kinase has several established inhibitors that have shown great potential in retarding the growth of tumors.
Over the past decade, several inhibitors have been identified that target specific protein kinases, and these inhibitors have been developed into highly effective anti-cancer agents. One such example is imatinib mesylate (Gleevec®), a tyrosine kinase inhibitor marketed by Novartis that successfully treats chronic myeloid leukemia and generates over $3.7 billion/year in revenue. There has been great difficulty in finding selective kinase inhibitors, and presently fewer than 15 have been approved by the FDA.